CN100498247C - Metal coated nanocrystalline silicon as an active surface enhanced raman spectroscopy (SERS) substrate - Google Patents

Abstract

The disclosed methods and devices relate to Raman spectroscopy utilizing metal-coated nanocrystalline porous silicon substrates. Porous silicon substrates can be formed by cationic etching in dilute hydrofluoric acid. Thin layers of Raman active metals, such as gold or silver, can be coated onto the porous silicon by using cathodic electromigration or any known technique. In certain aspects, metal-coated porous silicon substrates include plasma oxidized, dipped, and decomposed porous silicon substrates. Metal-coated substrates provide an extended, metal-rich environment for SERS, SERBS, Ultra-Raman and/or CARS Raman spectroscopy. In certain aspects, metal nanoparticles can also be added to metal-coated substrates to further enhance the Raman signal. Using the methods and devices of the present disclosure, Raman spectroscopy can be used to detect, identify and/or quantify various analytes. In some disclosed methods, Raman spectroscopy can be used to detect nucleotides, purines or pyrimidines at the single molecule level.

Description

Strengthen the nanocrystal silicon that is coated with metal of Raman spectroscopy (SERS) matrix as active surface

Related application

[0001] the application is the part continuation application of submitting on June 12nd, 2002 in aesthetic state patented claim 10/171,357, and the part continuation application of submitting on February 18th, 2003 in aesthetic state patented claim 10/368,976.

Technical field

[0002] this method and device relate to the Molecular Detection of utilizing Raman spectroscopy and/or the field of sign.More particularly, this method and device relate to the porous matrix that is coated with metal, and it is used for surface enhanced Raman spectroscopy (SERS), surface enhanced resonant raman spectroscopy (SERRS), super Raman and/or coherent antistockes Raman spectroscopy art (CARS).

Background technology

[0003] prove a task of difficulty to carrying out sensitivity from biological individual molecule and detecting accurately and/or identify, but they has extensive and potential application in medical diagnosis, pathology, toxicology, environmental sampling, chemical analysis, medical jurisprudence and other many fields with other samples.Use Raman spectroscopy and/or surface plasma body resonant vibration have been attempted, to realize this target.When light passed through tangible medium, the light of some was by its original direction transformation direction, and this is the phenomenon that is referred to as Raman scattering.Some scattered lights are different with original exciting light on frequency, and this is to arrive higher energy state because light is absorbed with electron excitation, and issues the cause of third contact of a total solar or lunar eclipse emission subsequently at different wave length.The wavelength of Raman emission spectrum is the chemical composition of extinction molecule in the sample and the sign of structure, and the intensity of light scattering depends on the concentration of molecule in the sample.

[0004] it is low-down the interactional probability of Raman taking place between the individual molecule in excitation beam and sample, and this causes the Raman analysis sensitivity low and use limited.Observe, it is the Raman scattering of 6 to 7 orders of magnitude that near the molecule coarse silver surface demonstrates enhancing.This surface enhanced Raman spectroscopy (SERS) effect and plasma resonance have necessarily gets in touch, wherein, because the collective of the conduction electron in metal coupling (collective coupling), metal nanoparticle demonstrates significant optical resonance to the electromagnetic radiation of incident.In fact, the nano particle of gold, silver, copper and some other metal can serve as miniature " antenna ", strengthens the local effect of electromagnetic radiation.Near these particles molecule demonstrates much bigger sensitivity in Raman spectrum analysis.

[0005] attempted removing to develop SERS to be used for Molecular Detection and analysis; this is normally by coating metal nano particle on the surface of matrix (substrate) or prepare coarse metal film, sample is imposed on to carry out on the matrix of metal coating then.Yet the quantity that can deposit to the metallic particles on the plane surface is limited, thereby for using this type of surperficial SERS and relevant Raman technology, will produce low relatively enhancer (enhancement factor).Needs to the manufacture method of SERS active matrix with high-density metal particle more and the device that comprises this kind matrix just produce thus.

The accompanying drawing summary

[0006] Xia Mian accompanying drawing constitutes the part of this instructions, and they are introduced instructions is in order to further specify some aspect of claimed method and apparatus.By with reference to wherein one or more accompanying drawings, and be combined in the detailed description that this provides, these method and apparatus can better be understood.

[0007] Fig. 1 illustrates exemplary device 100 (not drawn on scale) and the method that is used to produce porous silicon matrix 110.

[0008] Fig. 2 illustrates the exemplary method of the porous silicon matrix 240 that is used for the production washing.

[0009] Fig. 3 illustrates exemplary device 300 and method, and it is used for detecting and/or identification of analytes by the SERS active matrix 340 that uses washing.

[0010] Fig. 4 illustrates the exemplary method of the porous silicon matrix that is used for the production washing, comprises the thermally decomposable metal salt solusion.Fig. 4 A shows porous silicon matrix.Fig. 4 B illustrates silicon oxidation, for example by the plasma oxidation effect, forms silicon dioxide layer.The porous silicon that Fig. 4 C shows oxidation is immersed in the metal salt solution, in liquor argenti nitratis ophthalmicus.Fig. 4 D illustrates and removes excessive metal salt solution.Fig. 4 E shows solution is carried out drying, so that form the slaine thin layer of doing on porous silicon matrix.The slaine that Fig. 4 F illustrates doing carries out thermal decomposition, is covered in the even metal level of porous silicon matrix with formation.

[0011] Fig. 5 illustrates the uniform deposition of exemplary metal (silver) on porous silicon matrix, and this can obtain by the method for thermal decomposition.(A) the silver-colored depth section (Silver depth profile) of the nano-structure porous silicon of handling with traditional diffusion restriction immersion plating (diffusion limited immersion plating).(B) plasma oxidation that evenly applies with silver, dip and decompose (plasma-oxidized, dip and decomposed, PODD) the silver-colored depth section of porous silicon matrix.

[0012] Fig. 6 shows exemplary analyte, and the Surface enhanced raman spectroscopy of rhodamine 6G (R6G) dye molecule, is dipped and decomposed (PODD) porous silicon matrix and obtain its plasma oxidation by utilizing silver evenly to apply.PODD matrix gets by the method preparation of Fig. 4.Locate to excite at 785nm (nanometer), the R6G solution of 114 μ M is used to SERS (surface enhanced Raman spectroscopy).Fig. 6 shows the SERS emission spectrum with the matrix acquisition of the PODD silver coating of different porositys (porosities).Various spectrum is obtained, and by average porosity, the curve from the curve of below to the top is followed successively by 52%, 55%, 65%, 70% and 77%.

[0013] Fig. 7 shows the SERS detection of 90 μ M adenine solution of the nanoporous silicon matrix acquisition that utilizes washing, and the nanoporous silicon matrix of described washing makes according to the method for Fig. 4.

[0014] Fig. 8 shows the deoxyadenosine triphosphate solution (curve of top) of the fluorescein covalent labeling of 500nM and the comparison SERS spectrum of unlabelled dATP solution (curve of below).(Indianapolis IN) obtains this dATP-fluorescein from Roche Applied Science.In fluorescein-labeled dATP, detecting the SERS signal increases consumingly.

The description of exemplary embodiment

[0015] by utilizing surface enhanced Raman spectroscopy (SERS), surface enhanced resonant raman spectroscopy (SERRS) and/or coherent antistockes Raman spectroscopy art (CARS), method and apparatus of the present disclosure can be used to the detection and/or the evaluation of analyte.Compared with existing technology, method and apparatus of the present disclosure provides the metallic particles density with increase, better metal deposition uniformity and darker SERS to strengthen the SERS active matrix in territory (field), and this makes it possible to more effectively carry out the Raman detection and/or the evaluation of analyte.

[0016] Yi Qian the SERS detection method that is used for various analytes has been used the colloidal metal particle, and such as the silver nano-grain of assembling, it generally is applied on matrix and/or the holder (as United States Patent (USP) 5,306,403; 6,149,868; 6,174,677; 6,376,177).Yet, increase by 10 although make this being furnished with the time SERS detect ⁶To 10 ⁸Sensitivity, but they can't carry out the Single Molecule Detection of little analyte such as nucleotide, as disclosed herein.In the colloidal solid aggregation, the sensitivity of Raman detection strengthens and is inhomogeneous, depends on the existence of " focus " to a great extent.The physical arrangement of this kind focus, when the enhancing of sensitivity occurring and the distance range of nano particle, and the spatial relationship between aggregation nano particle and the analyte is not all also characterized when allowing sensitivity to occur strengthening.Further, the nano particle of gathering may be unstable in solution, and this can produce adverse influence to the repeatability that the single molecule analysis quality testing is surveyed.This method and device provide stabile microenvironment for SERS detects, and therein, the physical configuration of the metal matrix of Raman active and density can be controlled exactly, and this makes, and analyte can carry out repeatably at solution, sensitive and detection accurately.

[0017] following detailed comprises many details, so that understanding more completely claimed method and apparatus is provided.Yet it will be apparent for a person skilled in the art that does not have these details, and this method and/or device also can be implemented.In other examples, the device that this area has been known, method, program and each component are not described in detail in this article.

Definition

[0018] as used herein, " one " or " one " can refer to one or more than project.

[0019] as used herein, term " analyte (analyte) " refers to interested detected and/or any atom, chemical substance, molecule, compound, composition or the aggregation identified.The non-limitative example of analyte comprises: amino acid, peptide, polypeptide, protein, glycoprotein, lipoprotein, nucleosides, nucleotide, oligonucleotides, nucleic acid, sugar, carbohydrates, oligosaccharides, polysaccharide, fatty acid, lipid, hormone, metabolin, cell factor, chemotactic factor (CF), acceptor, neurotransmitter, antigen, anaphylactogen, antibody, substrate, metabolin, co-factor, inhibitor, medicament, medicine, nutrients, prion, toxin, poisonous substance, explosive, pesticide, chemical warfare reagent, the biohazard agent, radioactive isotope, vitamin, heterocyclic aromatic compound, carcinogen, mutagen, anesthetic, amphetamine, barbiturate, psychedelic, waste product and/or pollutant.Analyte can be a mark in addition not, maybe can carry out mark with one or more Raman labels things, and is as mentioned below.

[0020] be used in this article refer to can be in conjunction with any molecule of one or more target analytes for the molecule of " catching (capture) ".The non-limitative example of " catching " molecule comprises: antibody, antibody fragment, genetically engineered antibody, single-chain antibody, receptor protein, in conjunction with albumen, enzyme, inhibitor protein, agglutinin, cell adhesion albumen, oligonucleotides, polynucleotide, nucleic acid and fit.

[0021] as used herein, term " nanocrystal silicon (nanocrystalline silicon) " refers to comprise the silicon of the silicon crystal of nano-scale, and general size is in 1 to 100 nanometer (nm) scope." porous silicon (poroussilicon) " refers to etched or carries out other and handle, thus the silicon of formation porous structure.

[0022] as used herein, has interaction on the function between two or more unit of term " effectively connect (operably coupled) " finger device and system.For example, obtain, handle, preserve and/or transmit by the detected Raman signal of detecting device if calculate function, then this Raman detector is " effectively to connect " to this computing machine.

Porous matrix

[0023] some method disclosed herein relates to the coating porous matrix of the conforming layer with one or more metals such as Raman active metal.Although porous matrix disclosed herein is a porous silicon matrix, yet claimed theme is not restricted to those embodiment.Any porous matrix that can tolerate the heat application can be used in method disclosed herein, system and/or the device.The application that is heated to about 300 ℃, 400 ℃, 500 ℃, 600 ℃, 700 ℃, 800 ℃, 900 ℃ or 1000 ℃ can be considered.Porous matrix can be rigid, maybe can be flexible.Operable various porous matrix is known, including, but not limited to porous silicon, porous polycrystalline silicon, porous metals grid and porous aluminum.The exemplary method of preparation porous matrix will be disclosed in will further describing below.

[0024] porous polycrystalline silicon matrix can get (as United States Patent (USP) 6,249,080 and 6,478,974) with known technology preparation.For example, by using low-pressure chemical vapor deposition (LPCVD), porous polycrystalline silicon layer can be formed on the semiconductor substrate.The LPCVD condition can comprise, for example, and about 20 Pascals' pressure, about 640 ℃ temperature and the silane air-flow (United States Patent (USP) 6,249,080) of about 600sccm (standard cubic centimeter).For example, utilize the electrochemistry anodic oxidation that HF (hydrofluorite) carries out or utilize nitric acid and chemical method for etching that hydrofluorite carries out by use, polysilicon layer can be etched, so that it has poriness (United States Patent (USP) 6,478,974).Usually, the thickness limits of the porous polycrystalline silicon layer that forms by this class technology is at about 1 μ m (micron) or littler.On the contrary, porous silicon can be etched in the whole thickness of bulk silicon wafer, and its thickness is generally about 500 μ m.

[0025] porous aluminum matrix also can utilize known technology to prepare (as Cai et al., Nanotechnology13:627,2002; Varghese et al., J Mater.Res.17:1162-1171,2002).For example, use the auxiliary self-assembly process (electrochemical-assisted self-assembly process) of galvanochemistry, the nano-porous alumina film can be made (Cai et al., 2002) on silicon or silicon dioxide.Can carry out thermal annealing to porous aluminium, to improve its homogeneity (Cai et al., 2002).Alternately, the thin layer of solid aluminium can carry out electrochemical anodic oxidation in oxalic acid and/or sulfuric acid lean solution, to produce nanoporous aluminium film (Varghese et al., 2002).Example disclosed herein is not restrictive, can use the heat-resisting porous matrix of any kind.Use method disclosed herein, this type of porous matrix can flood such as silver equably with one or more metals.

Nanocrystalline porous silicon (Nanocrystalline Porous Silicon)

Nanocrystal silicon (Nanocrystalline Silicon)

[0026] some exemplary device disclosed herein can comprise one or more layers nanocrystal silicon.The whole bag of tricks of producing nanocrystal silicon is known in the art (as Petrova-Koch et al., ＂ Rapid-thermal-oxidized porous silicon-the superior photoluminescent Si, " Appl.Phys.Lett.61:943,1992; Edelberg, et al., ＂ Visible luminescence from nanocrystallinesilicon films produced by plasma enhanced chemical vapor deposition, ＂ Appl.Phys.Lett., 68:1415-1417,1996; Schoenfeld, et al., ＂ Formation of Si quantum dots innanocrystalline silicon, ＂ Proc.7th Int.Conf.on Modulated Semiconductor Structures, Madrid, pp.605-608,1995; Zhao, et al., ＂ Nanocrystalline Si:a material constructed bySi quantum dots, ＂ 1st Int.Conf.on Low Dimensional Structures and Devices, Singapore, pp.467-471,1995; Lutzen et al., Structural characteristics of ultrathinnanocrystalline silicon films formed by annealing amorphous silicon, J.Vac.Sci.Technology B 16:2802-05,1998; United States Patent (USP) 5,770,022; 5,994,164; 6,268,041; 6,294,442; 6,300,193).The method that method and apparatus disclosed herein is not produced nanocrystal silicon limits, and what should be thought of is, any known method can be used.

[0027] the non-restrictive illustrative method of production nanocrystal silicon comprises: silicon (Si) is implanted the oxide and the annealing of being rich in silicon; Carry out solid-phase crystallization with the metal nucleating agent; Chemical vapor deposition; PECVD (plasma enhanced chemical vapor deposition); Gas evaporation; Vapour-phase pyrolysis; Gas phase photolysis (gas phase photopyrolysis); The chemical etching method; The plasma decomposes of silane and polysilane; The high-pressure liquid phase oxidation-reduction reaction; The short annealing of amorphous silicon layer; With LPCVD (low-pressure chemical vapor deposition) deposited amorphous silicon layer, carry out RTA (rapid thermal annealing) circulation then; Use the arc plasma deposition of silicon anode and laser ablation (United States Patent (USP) 5,770,022 of silicon; 5,994,164; 6,268,041; 6,294,442; 6,300,193).Depend on technology, be of a size of 1 to 100nm or bigger Si crystal, can be used as thin layer and be formed on the chip, as the layer that separates and/or as the crystal of assembling.In some method and apparatus, can use thin layer, this thin layer comprises the nanocrystal silicon that is attached to hypothallus.

[0028] is contemplated that, in claimed method and apparatus, can uses nanocrystal silicon.Yet this method and device do not limit the composition of original material, in alternative method and device, be contemplated that, other materials also can be utilized, and unique requirement is that this material must can form porous matrix, and this porous matrix can apply with Raman sensitivity metal.

[0029] hole dimension in the size of silicon crystal and/or shape and/or the porous silicon can be selected within the predetermined scope, for example, so that optimize the plasma resonance frequency (referring to United States Patent (USP) 6,344,272) of the porous silicon that is coated with metal.Be coated on the metal layer thickness of porous silicon by control, the plasma resonance frequency can be adjusted (United States Patent (USP) 6,344,272).The technology of size that is used to control the silicon crystal of nanoscale is known (as United States Patent (USP) 5,994,164 and 6,294,442).

Porous silicon

[0030] some claimed method and apparatus relates to the matrix of using the washing with Raman active.This matrix can comprise nanocrystalline porous silicon.As mentioned above, this matrix is not limited to pure silicon, but can comprise silicon nitride, germanium and/or become known for other material of chip manufacturing.Other a spot of materials also can exist, such as metal nucleating agent and/or alloy.Unique requirement is, this host material must can form porous matrix, and this porous matrix can be used the washing of Raman sensitivity.Porous silicon has up to 783m ²/ cm ³High surface area, this provides very large surface for the surface enhanced Raman spectroscopy technology.

[0031],, found porous silicon by in dilute hydrochloric acid solution, silicon being carried out electropolishing in late period the 1950's.The road by in electrochemical cell, with diluted hydrofluoric acid (HF) etching silicon matrix, can produce porous silicon as known in the art.In some cases, silicon at first can be under low current density (current densities), and is etched in HF.After initial hole formed, silicon shifts out from electrochemical cell, and was etched in very rare HF, so that the hole that forms in electrochemical cell broadens.Depend on whether silicon is doped, the type of alloy and doping level, the composition of silicon matrix also can influence hole dimension.Doping is known in the art for the influence of silicon hole dimension.In order to detect and/or identify big biomolecule, can select the hole dimension of about 2nm to 100 or 200nm.Also can select the orientation (orientation) of hole in porous silicon.For example, etched 1,0,0 crystal structure will have the hole with the perpendicular orientation of this crystal, and 1,1,1 or 1,1,0 crystal structure will have the hole with crystal axis diagonal angle orientation.Crystal structure is known in the art to the influence of hole orientation.Crystal is formed and poriness also can be conditioned, to change the optical characteristics of porous silicon, so that strengthen Raman signal and reduce background interference.The optical characteristics of porous silicon is known in the art (as Cullis et al., J.Appl.Phys.82:909-965,1997; Collins et al., Physics Today 50:24-31,1997).

[0032] Fig. 1 shows the non-limitative example of the method and apparatus 100 that is used to produce porous silicon matrix.Silicon wafer 110 is placed in the electrochemical cell 120, and electrochemical cell 120 comprises inert material, such as

Wafer 110 is connected to the positive pole of constant current source 130, thereby forms the anode 110 of electrochemical cell 120.The negative pole of constant current source 130 is connected to negative electrode 140, such as platinum cathode 140.Electrochemical cell 120 can charge into the rare dielectric solution 150 of ethanol of HF.Alternately, HF150 can be dissolved in other alcohol known in the art and/or the surfactant, such as pentane or hexane.Computing machine can effectively be connected to constant current source 130, to regulate the time of electric current, voltage and/or chemical etching.The silicon wafer 110 that is exposed in the HF dielectric solution 150 in the electrochemical cell 120 is etched, thereby forms porous silicon matrix 110.Road as known in the art, by regulating the concentration of the HF 150 in anodised time and/or current density and the dielectric solution, the porous degree of the thickness of porous silicon layer and silicon can Be Controlled (as United States Patent (USP) 6,358,815).

[0033] by applying such as poly-methyl-methacrylate with any known resist compound, the part of silicon wafer 110 can exempt from HF 150 etched destructions.Lithographic process can be used for the part through selecting of silicon wafer 110 is exposed to HF 150 etchings such as photolithography, and this is known in the art.Selective etch can be used for controlling the size and dimension of the porous Si chamber that will be applied to Raman spectroscopy.For some application, can use the porous silicon chamber of the about 1 μ m (micron) of diameter.In other are used, can use porous silicon ditch or the passage of the about 1 μ m of width.The size in porous silicon chamber is not restrictive, can understand like this, can use the porous silicon chamber of virtually any size or shape.For example, can use the chamber size of 1 μ m, be equipped with the optical excited laser that is of a size of 1 μ m.

[0034] for producing porous silicon matrix, above disclosed exemplary method be not restrictive, can understand like this, any method known in the art can be used.The non-limitative example that is used to make the method for porous silicon matrix comprises: the etching anode method of silicon wafer; Electroplate; And the material of depositing silicon/oxygen, carry out in check annealing then; (Canham for example, ＂ Silicon quantum wire array fabrication byelectrochemical and chemical dissolution of wafers, ＂ Appl.Phys.Lett.57:1046,1990; United States Patent (USP) 5,561,304; 6,153,489; 6,171,945; 6,322,895; 6,358,613; 6,358,815; 6,359,276).Porous silicon layer may be attached to one or more supporting layers, such as piece silicon (bulk silicon), quartz, glass and/or plastics.Can use etching stopping layer (etch stop layer),, control etched depth such as silicon nitride.Can use known manufacturing method of chip, porous silicon layer is integrated in the semi-conductor chip.The porous silicon chamber that is coated with metal can be configured to the part of integrated chip, is connected in various passages, microchannel, nanochannel, miniflow attitude passage, reaction chamber etc.Alternately, the porous silicon chamber that is coated with metal can cut down from silicon wafer, and is integrated in chip and/or other devices.

[0035] be appreciated that and can carry out other modifications porous silicon matrix, this or before metallizing, carry out, perhaps after metallizing, carry out.For example, after etching, use methods known in the art, porous silicon matrix is oxidized into monox and/or silicon dioxide.Oxidation can be used to, and for example, increases the physical strength and the stability of porous silicon matrix.Alternately, the silicon matrix that is coated with metal can further carry out etching, to remove silicon materials, forms metal shell, and this metal shell can be a hollow, maybe can charge into other material, such as additional Raman active metal.

The washing of porous silicon

[0036] utilize any method known in the art, porous silicon matrix can be coated with Raman active metal, as gold, silver, platinum, copper or aluminium.Nonrestrictive exemplary method comprises plating; The cathodic electricity migration; Evaporation of metal or spraying plating; Use crystal seed to come catalysis to electroplate (that is it is gold-plated, to use copper/nickel crystal seed); Ion is implanted; Diffusion; Or be used for known in the art any method that thin metal layer is coated onto on the silicon matrix (is seen, Lopez andFauchet for example, ＂ Erbium emission form porous silicon one-dimensional photonic band gapstructures, ＂ Appl.Phys.Lett.77:3704-6,2000; As United States Patent (USP) 5,561,304; 6,171,945; 6,359,276).Other non-limitative examples of metallizing comprise that non-electrical coating is (as Gole et al., ＂ Patterned metallization of porous silicon from electroless solution for directelectrical contact, ＂ J.Electrochem.Soc.147:3785,2000).The composition of metal level and/or thickness can Be Controlled, are coated with the plasma resonance frequency of the porous silicon of metal with optimization.

[0037] the Raman active matrix that is used for detection of analytes can comprise the nanocrystalline porous silicon matrix that is coated with metal, immobilized metallic colloid, such as silver or gold nano grain, be applied on the dissimilar matrix, and/or immobilized metallic colloid is coated on the nanocrystalline porous silicon matrix of washing.The latter's composition will have very highdensity Raman active metal, be equipped with relatively little passage, so that the analyte in the solution enters in the matrix.Although this is for big analyte molecule, not too favourable such as protein and nucleic acid, it can offer little analyte, such as nucleotide or better sensitivity of amino acid and detectability.Metallic colloid can be the form of nano particle, and is as described below.

Know that [0038] arsenic-anodised porous silicon can serve as the gentle reductive agent of metallic ion, thereby cause the spontaneous immersion coating of metal on the porous region upper surface, and the blind hole opening.Therefore, use the standard method of metal impregnation, be difficult to obtain the metal depth section of homogeneous, keep the open state of porous surface simultaneously.Between the blind hole and the metal piercing degree of depth conflict is not being arranged, it can carry out description below.Obtain better metal depth section, the metallic ion of high concentration is necessary.Yet because the cause of the thick metal film deposition that is produced by the reaction of spontaneous dipping coating, the metal salt solution that is exposed to high concentration will make bore closure.In order to keep the open state in hole, GOLD FROM PLATING SOLUTION belongs to the concentration of ion must be lower.Yet this makes the length of penetration deficiency, and has reduced the quantity of metal deposition.By method disclosed herein, this problem is able to be solved, and its permission obtains more even metal deposition, and the hole is not closed simultaneously.

Utilize the thermal decomposition of slaine to carry out washing

[0039] as described in Figure 4, by comprising the method for thermally decomposable metal salt deposit 440, porous silicon matrix 410 can be used metal 450, applies equably such as Raman sensitivity metal 450.Metal 450 can be silver, gold or other Raman active metal.Porous silicon matrix 410 (Fig. 4 A) can, for example by top disclosed obtained like that.In order to prevent precocious metal 450 deposition and hole closed phenomenons, the superficial layer of silicon can be oxidized to silicon dioxide 420 (Fig. 4 B), for example finishes by chemical oxidation or plasma oxidation.By stablizing porous silicon surface 420, this oxidation has prevented spontaneous immersion plating.If do not carry out oxidation, the silver-colored kation of positively charged can participate in redox reaction with unoxidized silicon 410, thereby causes spontaneous silver metal 450 depositions.

[0040] carry out after the oxidation, porous silicon matrix 410 can be used metal salt solution 430, such as the silver nitrate (AgNO of 1M ₃) solution-wet (Fig. 4 C).In non-limitative example, the porous silicon matrix 410 of oxidation is dipped in the liquor argenti nitratis ophthalmicus 430, and the time is 20 minutes, uses liquor argenti nitratis ophthalmicus 430 wetting fully up to the hole.By for example nitrogen rifle seasoning (nitrogen gun drying), excessive metal salt solution is removed (Fig. 4 D).By for example be heated to 100 ℃ 20 minutes, be trapped in solution 430 in the hole can be dried (Fig. 4 E).At this moment, solvent evaporates, and the thin layer of the nitric acid silver salt of doing 440 is deposited on the surface of porous silicon 410.By for example in the environmental pressure stove, be heated to 500 ℃ 30 minutes, the salt of doing 440 can be thermal decomposited (Fig. 4 F).Under the temperature that is higher than about 573 ° of K (about 300 ℃), the reaction of formula 1 is spontaneous to be carried out.According to formula 1, nitrate ion is converted to nitrogen dioxide gas, thereby forms the uniform deposition layer (Fig. 4 F) of the argent 450 that is coated on porous silicon matrix 410.Although nitrogen dioxide has been used as the photoetch agent, under the condition of disclosure method, it is etch silicon dioxide layer 420 not.

AgNO ₃→ Ag (s)+NO ₂(gas)+1/2 O ₂(gas) (1)

[0041] by for example changing the concentration of metal salt solution 430, the thickness of the metal level 450 of deposition can Be Controlled.The thickness that depends on the metal level 450 that will deposit, the concentration of salt solusion 430 can change in the scope of broad, is about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.25,1.5,1.75,2.0,2.5,3.0,3.5,4.0,4.5 to 5.0M (molar).Although exemplary method is to utilize silver-colored solution 430, this method is not limited to depositing silver 450, but can comprise any known metal 450, includes, but are not limited to Raman active metal, such as gold, copper, platinum, aluminium etc.This method does not limit the type of the salt of use yet.The anion species that is used to form slaine can be such, and it is converted into gaseous matter during thermal decomposition process, and is purged, such as nitrate radical or sulfate ion.Yet, in other replacement schemes, can have no restrictedly to use any anionic species.

Utilize miniflow attitude dipping to carry out washing

[0042] in alternative method, by utilizing miniflow attitude dipping (microfluidic impregnation), perforated membrane such as porous silicon film, can be used washing.In exemplary method, porous silicon film can be as top obtained disclosed.Porous silicon layer can and be suspended in the solution by electropolishing.The film of electropolishing can be inserted in the one or more solvent containers and the miniflow attitude passage between the waste canister that connect by cross path (cross-paths).This miniflow attitude passage can produce by any method known in the art, such as the lithography technique or the photolithography of carrying out micro shaping, standard with PDMS (dimethyl silicone polymer), and to the etching technique of various chip materials (as Duffy et al., Anal.Chem.70:4974-84,1998).Porous silicon film can be integrated in the miniflow attitude system of any kind.The miniflow attitude system that is integrated with porous silicon film can be used for various widely application, and it relates to the analysis and/or the separation of polymer molecule, and described polymkeric substance includes but not limited to protein and nucleic acid.The method that is used for micron and/or nanoscale manufacturing is known in the art, as following institute in greater detail.

[0043] metal salt solution can be conducted through solvent container such as liquor argenti nitratis ophthalmicus, and allows its porous silicon film of flowing through, and arrives waste canister.As shown in Equation 2, spontaneous reaction can take place.

Ag ⁺(aq.)+Si (surface)+2H ₂O (liquid) → Ag (solid)+H ₂(gas)+SiO ₂(surface)+2H ⁺(2)

[0044] disclosed suc as formula 2, in redox reaction, moisture metallic solution spontaneously reacts with porous silicon surface, thereby produces the metallic coating of deposition on porous silicon.Belong to the concentration of salt, speed, the temperature of the miniflow attitude passage of flowing through by adjusting GOLD FROM PLATING SOLUTION, and/or the duration of flow of solution when film, the thickness of metallic coating can Be Controlled.The technology of controlling such plating reaction is known in the art.

[0045] this method is not limited to silver-colored solution, also can implement with other metal salt solutions, includes but not limited to Raman active metal, such as gold, platinum, aluminium, copper etc.In other alternatives, comprise a plurality of solvent containers of different metal plating bath by use, the metals that two or more are different are plated on the porous silicon film.One or more containers can comprise wash solution, to remove excessive metal plating liquid.With the coating that multiple metal carries out, can be used to control electricity, light and/or the Raman surface characteristic of the porous silicon film of washing, such as, the surface of Raman signal strengthen degree, with the distance on the surface that resonance takes place, the wavelength coverage of resonance Raman etc.

[0046] disclosed method causes producing the porous silicon film that is coated with metal that is integrated into miniflow attitude passage.A kind of integrated chip like this can directly be integrated in the Raman detection system.One or more samples that contain target molecule under a cloud can be loaded in the corresponding solvent container.Sample can be conducted through miniflow attitude passage by passage, enters the film that is coated with metal.In case enter in the film, the target molecule light source that can be excited excites such as laser.The Raman signal of emission can be detected by Raman detector, describes in detail more as following.In case after analyzed, sample can be moved in the waste canister, film is washed, and carries out the analysis of next sample.The Raman detection system can integrate various assembly known in the art, such as Raman detector and excitation source, maybe can comprise the customizable component of design, and these assemblies will be fully integratible in the system, to optimize the Raman detection of analyte.

Nano particle

[0047] Raman active metal particles such as gold or silver nano-grain, be introduced in the porous silicon matrix that is coated with metal, with further enhancing Raman signal.The nano particle of diameter between 1nm and 2 μ m can be used.Alternately, can consider the nano particle of diameter between 2nm to 1 μ m, 5nm to 500nm, 10nm to 200nm, 20nm to 100nm, 30nm to 80nm, 40nm to 70nm or 50mn to 60nm.Also can consider mean diameter 10 to 50nm, 50 nano particles to 100nm or about 100nm.The size of nano particle depends on the bore dia in the porous silicon that is coated with metal, can select so that nano particle just in time is contained in the inside, hole this size.Although Any shape or erose nano particle can be used, nano particle can be almost spherical in shape.The method for preparing nano particle is known (as United States Patent (USP) 6,054,495; 6,127,12; 6,149,868; Lee and Meisel, J.Phys.Chem.86:3391-3395,1982).Nano particle also can produce (Jin et al., ＂ Photoinduced conversion of silver nanospheres to nanoprisms, ＂ Science294:1901,2001) in the mode of nanoprisms.Nano particle can obtain (Nanoprobes Inc. for example, Yaphank, NY from the commercial channel; Polysciences, Inc., Warrington, PA).

[0048] nano particle can be the random aggregation body (colloidal nano particle) of nano particle.Can be alternatively, nano particle can be crosslinked, to produce specific aggregates of nanoparticles, such as dimer, tripolymer, the tetramer or other aggregations.Can use the aggregation heterogeneous mixture that constitutes by a group aggregates of nanoparticles different or homogeneity.By utilizing known technology, such as the super centrifugation technique in Sucrose gradient solutions, the aggregation that contains the nano particle (dimer, tripolymer etc.) of selected quantity can be by enrichment or purifying.

[0049] method of crosslinking nano particle be known in the art (referring to, Feldheim for example, ＂ Assembly ofmetal nanoparticle arrays using molecular bridges, ＂ The Electrochemical SocietyInterface, Fall, 2001, pp.22-25).Gold nano grain is known (Feldheim, 2001) with the reaction that is connected compound with terminal mercaptan or mercapto groups.Single connection compound can be derived with thiol group at its two ends.In case after the gold nano grain reaction, this connector will form the nano particle dimer, its length by connector is spaced apart.Alternately, have three, four or more the connector of polythiol group can be used to be attached to simultaneously (Feldheim, 2001) on a plurality of nano particles.With respect to connecting compound, use excessive nano particle can prevent to form how crosslinked and nanoparticle precipitate.The aggregation of silver nano-grain can form by standard synthetic method known in the art.

[0050] gold or silver nano-grain can be used derivatization silane, apply such as amino silane, 3-glycidoxypropyl trimethoxysilane (GOP) or TSL 8330 (APTS).The reactive group of silane end can be used to form the cross-linked aggregates of nano particle.What can expect is, the connection compound that uses almost can be any length, and scope is about 0.05,0.1,0.2,0.5,0.75,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,27,30,35,40,45,50,55,60,65,70,80,90 to 100nm or bigger length.Also can use the connector of different length.

[0051] before being attached to the connector compound, nano particle can be modified, to comprise various reactive groups.The nano particle of modifying can obtain from the commercial channel, such as

Nano particle, it is from Nanoprobes, Inc. (Yaphank, NY).

Nano particle can obtain by single or multiple maleimides, amine or other groups are attached on each nano particle.

Nano particle also has positively charged or electronegative form, with the convenient nano particle of handling in electric field.The nano particle of such modification may be attached on the various known connection compounds, with dimer, tripolymer or other aggregations that nano particle is provided.

[0052] type of employed connection compound is unrestricted, as long as it produces the little aggregation of the nano particle that can not precipitate in solution.For example, the connector group can comprise phenylacetylene polymkeric substance (Feldheim, 2001).Alternately, the connector group can comprise teflon, polyvinylpyrrolidone, polystyrene, polypropylene, polyacrylamide, tygon or other known polymkeric substance.Employed connection compound is not limited to polymkeric substance, also can comprise the molecule of other types, such as silane, alkane, derived silanes or the alkane of deriving.Especially, chemical constitution relatively simply connects compound, such as alkane or silane, can be used, to avoid interference the Raman signal that analyte sends.

Microelectromechanical systems (MEMS)

[0053] the porous silicon matrix of Raman active metal coating can be integrated in the bigger device and/or system.For example, this matrix can be integrated in the microelectromechanical systems (MEMS).MEMS is the integral system that comprises mechanical organ, sensor, actuator and electron device.All these assemblies can be made on common chip by known micro-processing technology, and described chip comprises matrix (for example, Voldman et al., Ann.Rev.Biomed.Eng.1:401-425,1999) silica-based or that be equal to.The sensor module of MEMS can be used to measurement mechanical, heat, biology, chemistry, optics and/or magnetic phenomenon.Electron device can be handled the information from sensor, and the control actuator assembly, such as pump, valve, well heater, refrigeratory, filtrator etc., thus the function of control MEMS.

[0054] electronic package of MEMS can use integrated circuit (IC) technology (as CMOS, Bipolar or BICMOS technology) manufacturing.They can use photoengraving known in the computer chip manufacturing and engraving method to come moulding.Micromechanical component can be with compatible " micromachined " the technology manufacturing, etch away to this process choice the silicon wafer of part or add new structural sheet, thereby form machinery and/or dynamo-electric assembly.

[0055] basic fundamental of making MEMS comprises: deposit film material on matrix, and apply at the film top by lithoprinting imaging or other known lithographic methods and to have sheltering of certain pattern, and the described film of etching optionally.The thickness of film can be in the scope of several nanometers to 100 micron.The deposition technique that uses can comprise chemical method, such as chemical vapor deposition (CVD), electro-deposition, epitaxial growth and thermal oxide, and physical method, such as physical vapor deposition (PVD) and casting.The method of making the nano-electromechanical system can be used in certain embodiments of the present invention (referring to, Craighead for example, Science 290:1532-36,2000).

[0056] the porous silicon matrix that is coated with metal can be connected to the various chambers that are full of fluid, such as miniflow attitude passage, nanochannel and/or microchannel.These assemblies in the device and other assemblies can form a single unit, and for example, the form of chip is as known in semi-conductor chip and/or microscopic capillary or miniflow attitude chip.Alternately, the porous silicon matrix that is coated with metal can be removed from silicon wafer, and is attached to other assemblies of equipment.Any material that becomes known for this type of chip can be applied to device disclosed herein, comprises silicon, silicon dioxide, silicon nitride, dimethyl silicone polymer (PDMS), polymethylmethacrylate (PMMA), plastics, glass, quartz etc.

[0057] technology that is produced in batches of chip is that computer chip manufacturing and/or microscopic capillary chip manufacturing field are known.This type of chip can be used any known method manufacturing in this area, such as lithoprinting and etching, laser ablation, injection molding, casting, molecular beam oriented growth, dip in a nanometer etching, chemical vapor deposition (CVD) processing, electron beam or focused ion beam technology or printing technology.The example of indefiniteness comprises: with material such as plastics or glass carry out conventional molding clearly on flowable, the optics; The photoengraving of silicon dioxide and dry etching; Use the polymethylmethacrylate resist and carry out electron beam lithography, make the aluminium mask on the silicon nitride matrix 140 form pattern, carry out active-ion-etch subsequently.The method that is used to make the nano-electromechanical system can be used to certain embodiments of the present invention (referring to, Craighead for example, Science 290:1532-36,2000).Various forms of little manufacturing chips can obtain from the commercial channel, for example Caliper Technologies Inc. (Mountain View, CA) and ACLARA BioSciences Inc. (Mountain View, CA).

[0058] device can be selected as partly or entirely that to excite the electromagnetic radiation with transmission frequency be transparent to being used for being in of Raman spectroscopy, such as other any material clearly on glass, silicon, quartz or the optics.For the chamber that is full of fluid that can be exposed to various biomolecule such as protein, peptide, nucleic acid, nucleotide and analog, the surface that is exposed to these molecules can be carried out modification by coating, so that for example hydrophobic surface is converted into hydrophilic surface, and/or reduce molecular adsorption to the surface.The finishing of common chip material such as glass, silicon, quartz and/or PDMS is (as United States Patent (USP) 6,263,286) known in the art.This type of modification can including, but not limited to, with commercially available capillary coating (Supelco, Bellafonte, PA), have the silane of various functional groups such as polyethylene oxide or acrylamide, or any other coating known in the art applies.

Raman spectroscopy

[0059] utilize surface enhanced Raman spectroscopy (SERS), surface enhanced resonant raman spectroscopy (SERRS) and/or coherent antistockes Raman spectroscopy art (CARS) to detect, disclosed mthods, systems and devices can be used to the detection and/or the evaluation of analyte.Compared with existing technology, mthods, systems and devices disclosed herein provide have increase and more even metal density and darker SERS strengthen the SERS active matrix of scope, this makes can more effectively carry out the Raman detection and/or the evaluation of analyte.

[0060] Yi Qian the SERS detection method that is used for various analytes has been used the colloidal metal particle, and such as the silver nano-grain of assembling, it generally is applied on matrix and/or the holder (as United States Patent (USP) 5,306,403; 6,149,868; 6,174,677; 6,376,177).Yet, increase by 10 although make this being furnished with the time SERS detect ⁶To 10 ⁸Sensitivity, but they can't carry out the Single Molecule Detection of little analyte such as nucleotide, as disclosed in this.In the colloidal solid aggregation, the sensitivity of Raman detection strengthens also inhomogeneous significantly, but depends on the existence of " focus " to a great extent.The physical arrangement of this kind focus, when the enhancing of sensitivity occurring and the distance range of nano particle, and the spatial relationship between aggregation nano particle and the analyte is not all also characterized when allowing sensitivity to occur strengthening.Further, the nano particle of gathering may be unstable in solution, and this can produce adverse influence to the repeatability that the single molecule analysis quality testing is surveyed.This method and device provide stabile microenvironment for SERS detects, and therein, the physical configuration of the metal matrix of Raman active and density can be controlled exactly, and this makes, and analyte can carry out repeatably at solution, sensitive and detection accurately.

The Raman detection instrument

[0061] by any known method of Raman spectroscopy, analyte can detected and/or evaluation.Raman active matrix can effectively be coupled on one or more Raman detection devices.The whole bag of tricks that utilizes Raman spectroscopy check and analysis thing is known in the art (as United States Patent (USP) 6,002,471; 6,040,191; 6,149,868; 6,174,677; 6,313,914).The various modification of surface enhanced Raman spectroscopy (SERS), surface enhanced resonant raman spectroscopy (SERRS), super Raman spectroscopy and coherent antistockes Raman spectroscopy art (CARS) are disclosed.In SERS and SERRS, for being attracted to roughened metal surface, such as silver, gold, platinum, copper or the lip-deep molecule of aluminium, the sensitivity of Raman detection is increased 10 ⁶Or more times (factor).

[0062] non-limitative example of Raman detection device is disclosed in United States Patent (USP) 6,002,471.Excitation beam is produced at 532nm wavelength place by frequency multiplication (frequency doubled) Nd:YAG laser instrument, and perhaps by frequency multiplication Ti: sapphire laser produces at 365nm wavelength place.Pulse laser beam or continuous laser beam can be used.Excitation beam is passed confocal optics and micro objective, is focused on Raman active matrix, and this Raman active matrix contains one or more analytes.Raman emission light from analyte is collected by micro objective and confocal optics, and is coupled to the monochromator that is used for spectrum disassociation (spectral dissociation).Confocal optics comprises the combination of dichroic filters, barrier filters, confocal pinhole, lens and reflective mirror, and they are used to reduce background signal.The full visual field optical device of standard can equally be used with confocal optics.Raman emission can detect with Raman detector, and described detecting device can comprise and be used to calculate the avalanche diode that joins with signal digitalized computing machine.

[0063] at United States Patent (USP) 5,306, in 403, alternative example of Raman detection device is disclosed, it comprises 1403 pairs-grating spectrophotometer of Spex Model, be furnished with gallium arsenide photomultiplier (RCA ModelC31034 or Burle Industries Model C3103402), it is operated with the single photon counting pattern.Excitaton source comprises the argon laser of 514.5nm spectrum line, and from SpectraPhysics, Model 166; (Innova 70, Coherent) with the krypton ion laser of 647.1nm spectrum line.

[0064] alternative excitaton source comprises the nitrogen laser (Laser Science Inc.) at 337nm place and He-Cd laser device (the Liconox) (United States Patent (USP) 6 at 325nm place, 174,677), light emitting diode, Nd:YLF laser instrument, and/or various ion laser and/or dye laser.Excitation beam can be carried out the spectrum purifying with bandpass filter (Corion), and can use 6X object lens (Newport, Model L6X) to focus on the Raman active matrix.By using Holographic Beam Splitter (Kaiser Optical Systems, Inc., Model HB 647-26N18), object lens can be used to excite analyte and collect Raman signal, thereby produce the right angle geometric relationship of the Raman signal of excitation beam and emission.(Kaiser Optical Systems Inc.) can be used to reduce rayleigh scattered radiation to holographic notch filters.Alternative Raman detector is including, but not limited to ISA HR-320 spectrograph, it is equipped with red reinforcement charge-coupled image sensor (red-enhanced intensified charge-coupleddevice, RE-ICCD) detection system (Princeton Instruments) that strengthens.The detector of other types also can be used, such as fourier transform spectrograph (based on the Michaelson interferometer), charge injection device (chargedinjection devices), photodiode array, InGaAs detector, electron multiplication CCD, enhanced type CCD and/or photo-transistor arrays.

[0065] Raman spectroscopy of any suitable form or structure or correlation technique known in the art can be used to the check and analysis thing, comprise, but be not limited to common Raman scattering, resonance Raman scattering, Surface enhanced raman spectroscopy, serrs, coherent antistockes Raman spectroscopy art (CARS), stimulated Raman scattering, anti-Raman spectroscopy, be excited acquired Raman spectroscopy (stimulated gain Ramanspectroscopy), hyper, the molecular optics laser detector (molecular optical laserexaminer, MOLE) or Raman microprobe or Raman microtechnic or the burnt Raman microspectrum of copolymerization art, three-dimensional or scanning Raman, the saturated spectroscopy of Raman, the time resolution resonance raman, Raman decoupling spectroscopy or ultraviolet-Raman microtechnic.

Raman labels

[0066] some method can relate to mark is attached on one or more analytes, detects them with convenient with the Raman detection device.The indefiniteness example that can be used for the mark of Raman spectroscopy comprises TRIT (the different mercaptan of tetramethylrhodamin), NBD (7-nitrobenzene-2-Evil-1, the 3-diazole), the Texas red, phthalic acid, terephthalic acid (TPA), m-phthalic acid, cresols is solid purple, the cresols royal purple, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, biotin, digoxin, 5-carboxyl-4 ', 5 '-two chloro-2 ', 7 '-the dimethoxy fluorescein, 5-carboxyl-2 ', 4 ', 5 ', 7 '-tetrachlorofluorescein, the 5-Fluoresceincarboxylic acid, 5-carboxyl rhodamine, 6-carboxyl rhodamine, the amino phthalocyanine of 6-carboxyl tetramethyl, azomethine, cyanines, xanthine, succinylfluoresceins, aminacrine, quantum dot, carbon nano-tube and fullerene.The Raman labels of these and other can obtain from the commercial channel (as MolecularProbes, Eugene.OR; Sigma Aldrich Chemical Co., St.Louis, MO), and/or synthetic with methods known in the art.

[0067] polycyclc aromatic compound can be used as Raman labels, and this is known in this area.Other marks that can be used for particular of the present invention comprise prussiate, mercaptan, chlorine, bromine, methyl, p and s group.Usage flag is (as a United States Patent (USP) 5,306,403 and 6,174,677) known in the art in Raman spectroscopy.The technician will recognize that employed Raman labels should be able to produce discernible Raman spectrum, and can be attached to or be connected to dissimilar analytes specifically.

[0068] mark can directly be attached to analyte, maybe can be attached to analyte by various connection compounds.The cross-linking reagent that is used for disclosure method is known in the art with being connected compound.The Raman labels that contains reactive group can obtain by the commercial channel (for example, Molecular Probes, Eugene, OR), reactive group wherein is designed to by the mode of covalency and other molecules, such as analyte response.The method that is used to prepare the analyte of mark is known (as United States Patent (USP) 4,962,037; 5,405,747; 6,136,543; 6,210,896).

Embodiment

Embodiment 1: the structure of Raman active matrix

The formation of nanocrystal silicon

[0069] exemplary method and the device that is used to form nanocrystalline porous silicon matrix 110 100 is illustrated in Fig. 1.The method that is used to make nanocrystalline porous silicon is (as United States Patent (USP) 6,017,773) known in the art.Nanocrystalline porous silicon layer can form with electrochemical method, and is disclosed as (Appl.Phys.Let.61:943,1992) such as Petrova-Koch.Depend on concrete application, before etching, silicon can be mixed by light or heavy p type or n type, to regulate the characteristic of porous silicon matrix 110.Monocrystal silicon can be the manufacturing of the known Czochralski method of people (as Http:// www.msil.ab.psiweb.com/ehglish/msilhist4-e.html).Silicon single crystal wafer 110 can be handled with the etching anode method, to form nanocrystalline porous silicon matrix 110 in rare HF/ ethanol 150.Alternately, can use the chemical method for etching that in the solution 150 of HF, nitric acid and water, carries out, and need not to carry out etching anode.

[0070] before etching, wafer can apply with poly-methyl-methacrylate resist or any other known resist compound.By utilizing the photolithographic techniques of standard, the pattern of nanocrystalline porous silicon matrix 110 can be formed.Nanocrystalline porous matrix 110 is the shape of (trench shaped), flute profile (channel shaped) or any other selection circular, ditch shape.A plurality of porous matrixes 110 can form on single silicon wafer 110, so that can be formed for the many samplings passage and/or the chamber of Raman analysis.Each sampling passage and/or chamber can effectively be connected to one or more Raman detector.

[0071] carry out after resist-coating and the lithography, wafer 110 is exposed in HF 150 solution that are in the about 15wt% to 50wt% in ethanol and/or the distilled water, and this solution is included in the electrochemical cell 120, this electrochemical cell 120 by

Constitute, disclosed as Fig. 1.Whole wafer 110 through resist-coating can be dipped into HF 150 solution.Alternately, for example use synthetic rubber washer, wafer 110 can be fixed in the electrochemical cell 120, and only Yi Bufen wafer 110 surfaces are exposed to HF 150 solution (United States Patent (USP) 6,322,895).In either case, wafer 110 is connected to the positive pole of constant current source 130 in the mode of electricity, to form the anode 110 of electrochemical cell 120.Platinum electrode can provide negative electrode 140 for this pond 120.In the dark, use 5 to 250 milliamperes/cm ²Oxidation current density, wafer 110 can be etched 5 seconds to 30 minutes, this depends on the porous degree of selection.In particular of the present invention, about vesicularity of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90% can be selected.Whether the road forms the type that porous silicon 110 needed oxidation current density can partly depend on employed silicon matrix 110 as known in the art, mixed by light or heavy p type or n type such as matrix 110.

[0072] use known chip fabrication techniques, nano particle porous silicon matrix 110 can be integrated into the MEMS device, and the MEMS device comprises various detectors, sensor, electrode, other electronic packages, mechanical actuator etc.Such manufacturing process can and/or be carried out before porous silicon matrix 110 forms and/or apply with Raman sensitivity metal afterwards.

Washing

[0073] as shown in Figure 2, use known technology, porous silicon 210 can pass through the electromigratory method of negative electrode, applies (Lopez and Fauchet, 2000) with metal 240.Although other metals 240 also can be used such as gold or platinum, for reaching the purpose of present embodiment, silver is used to washing 240.Clean porous silicon surface 210, and the electromigration method by Lopez and Fauchet (Appl.Phys.Lett.75:3989,1999), silver-doped 240.Porous silicon matrix 210 is exposed to the metal ion solution 220 that comprises silver-colored kation 230.Those skilled in the art will recognize that, can use any known technology that is used on porous silicon matrix 210, forming thin metallic coating 240.

Embodiment 2: utilize thermal decomposition that porous silicon is carried out washing

[0074] Fig. 4 illustrates metal 450 is impregnated into exemplary method in the nano-structure porous silicon equably.The surface of porous silicon is oxidized to silicon dioxide (Fig. 4 B).Metal salt solution is diffused in the porous matrix (Fig. 4 C), and be dried (Fig. 4 E).Dry slaine is thermal decomposited in the inside, hole, thereby forms even metal layer (Fig. 4 F).The oxidation of porous silicon surface makes that porous silicon can be wetted fully in metal salt solution, prevents spontaneous immersion coating simultaneously, spontaneous soak the coating that stagnates and will make the hole sealing.The slaine of doing is thermal decomposited in stove, and pure metal is deposited on the inwall of nano-pore.Can obtain uniform, the very thin metallic coating of nano-structure porous silicon, and can plugging hole, the phenomenon of plugging hole often is found in the standard method of metal penetration being gone into nano-structure porous silicon.The existing plating method also is that diffusion is upward limited, thereby forms uneven metal deposition, and uneven metal deposition can reduce the repeatability of detection of analytes, and the result depends on the position of analyte in being coated with the matrix of metal.

[0075] make the whole pore structure of washing need best dip time and high concentration of metal ions.Before being exposed to metal salt solution, oxidation to be carried out on the surface of porous silicon can be satisfied these needs, oxidation can be that chemical oxidation maybe can be plasma oxidation (Fig. 4 B).Oxidation prevents spontaneous immersion plating by the surface of stable hole.Therefore the porous silicon of oxidation can be dipped into the metal salt solution of high concentration, and can not cause hole closed phenomenon (Fig. 4 C).For example by nitrogen flushing gas, excessive metal salt solution can be removed (Fig. 4 D).Solvent evaporation is fallen, to increase the absorption (Fig. 4 E) of slaine on porous surface.Metal is thermal decomposited (Fig. 4 F), so that Raman active metal forms uniform deposition on the surface of porous silicon matrix.

[0076] in non-restrictive example, porous silicon matrix is formed by following method: carry out chemical etching in 15%HF solution, the crystalline silicon of doped with boron is exposed to 50mA/cm ²Electric current in.In Technics oxygen plasma chamber, adopt oxygen flow speed and 300W (watt) radio-frequency power of 50sccm (standard cubic centimeter), porous silicon matrix was carried out plasma oxidation 20 minutes, thereby form about 50 at hole surface

The silicon dioxide layer of (dust).Alternately, can in piranha solution, carry out the chemical oxidation effect (as Http:// www-device.eecs.Berkeley.edu/～daewon/labweek7.pdf).Silicon dioxide layer can hang key (dangling bond) by passivation silicon, thereby prevents immersion coating fast.

[0077] at room temperature, the porous silicon of oxidation is dipped in the AgNO of 1M ₃Solution 20 minutes is so that with liquor argenti nitratis ophthalmicus complete wetting hole.Excessive liquor argenti nitratis ophthalmicus is removed by nitrogen rifle seasoning, with the hole closure that prevents to be caused by excessive deposition of silver.By 100 ℃ of dryings 20 minutes, solvent is removed from remaining liquor argenti nitratis ophthalmicus.In this stage, all solvents are evaporated, and the nitric acid silver salt of doing is attracted on the surface in hole, forms observable brown on the surface of porous silicon.

[0078] in the environmental pressure stove, at 500 ℃, carried out thermal decomposition 30 minutes, thereby the nitric acid silver salt thermal decomposition that causes doing becomes silver metal.As shown in Figure 5, method disclosed herein makes the height that forms silver metal on the surface of porous silicon matrix deposit uniformly.Fig. 5 shows the silver-colored depth section of obtaining on nano-structure porous silicon, this is to measure and obtain with Rutherford backscattering spectral analysis.Will be at the AgNO of 1mM ₃In the solution, the silver-colored depth section (Fig. 5 A) of the nano-structure porous silicon that obtains with traditional diffusion limited immersion plating processing 2.5 minutes compares with the silver-colored depth section (Fig. 5 B) that the method with present embodiment obtains.As can be seen, compared with standard method, this method obtains deposition of silver highly uniformly, and has bigger length of penetration (Fig. 5 A and Fig. 5 B).This method produces the degree of depth and reaches deposition of silver (Fig. 5 B) about 10 μ m, uniform, and standard method produces the degree of depth less than deposition of silver about 3 μ m, extremely uneven (Fig. 5 A).By using scanning electron microscopy analysis, Rutherford backscattering data are revised, to record the actual (real) thickness of porous silicon layer.

[0079] will use the silver that this method obtains and the distribution of silicon to compare (Fig. 5 B), and can observe, deposition of silver is uniformly, reaches peaked position until silicon density.That is to say that the data presentation of Fig. 5 B deposits always uniformly extension to the bottom of porous silicon matrix mesopore with the metal that this method obtained.Clearly, use standard method (Fig. 5 A), arrive the bottom, hole very early before, the metal deposition just is through with.

Embodiment 3: the Raman detection of analyte

[0080] matrix 240,340 that applies by the formed Raman active metal of method as disclosed above can be integrated into and be used for detection of analytes, evaluation and/or quantitative device 300, as illustrated in Fig. 3.Matrix 240,340 can be integrated into, and for example in the flow cell 330, it is connected to inlet 320 and outlet 350 passages.Access road 320 can be connected to one or more other devices 310, such as sample injector 310 and/or reaction chamber 310.The analyte pond 330 that can circulate, and by Raman active matrix 340, there, they can be detected by Raman detection device 360.Pick-up unit 360 can comprise Raman detector 380 and light source 370, such as laser instrument 370.Laser instrument 370 can be launched excitation beam 390, the activation analysis thing, and cause the emission of Raman signal.Raman signal can detect by detected instrument 380.In certain embodiments of the invention, detector 380 can effectively be connected to computing machine 395, and data can be handled, analyze, preserve and/or transmit to computing machine 395, and these data are about being present in the data of the analyte in the sample.

[0081] in the exemplary embodiment that the present invention divides, excitation beam 390 is by titanium: sapphire laser 370 (Tsunami, Spectra-Physics) near infrared ray wavelength place (750-950nm), or produce at 785nm or 830nm place by aluminum gallium arsenide diode laser 370 (PI-ECL series, Process Instruments).Can use pulse laser beam 390 or continuous light beam 390.Excitation beam 390 is formed the conllinear geometric relationship by dichronic mirror (holographic notch filters of Kaiser Optical, perhaps, the interference filter of Chroma or Omega Optical) reflection with the light beam of collecting.Beam reflected 390 is passed micro objective (Nikon LU series), focuses on the Raman active matrix 240,340, has target analytes on this matrix.Raman diffused light from analyte is collected with same micro objective, passes dichronic mirror and reaches Raman detector 380.Raman detector 380 comprises condenser lens, spectrograph and array detector.Condenser lens is with the entrance slit of Raman diffused light focusing by spectrograph.This spectrograph (RoperScientific) comprises grating, and it disperses light according to wavelength.The light that disperses is imaged on the array detection instrument (the Back-illuminated Deep-depletionCCD camera of RoperScientific).This array detection instrument is connected to controller circuitry, and it is connected to again and is used on data shift and detector 380 functions are controlled the computing machine 160,395.

[0082] in various embodiments of the present invention, pick-up unit 360 can be with the sensitivity of height, detection, evaluation and/or quantitative many analytes, and even the detection of individual molecule and/or evaluation.In certain embodiments of the invention, analyte can comprise single nucleotide, and nucleotide can carry out Raman labels or not carry out mark.In other embodiments, one or more oligonucleotide probes can with or need not discernible Raman labels carry out mark, and can hybridize on the target nucleic acid in the sample.By with the oligonucleotide probe hybridization of complementation, and use the device 300 of Fig. 3 to carry out Raman detection, whether the existence of target nucleic acid can be instructed to out.Alternately, use the methods and apparatus disclosed 300, interested amino acid, peptide and/or protein can detected and/or evaluations.Those skilled in the art will recognize that; method and apparatus 300 does not limit can be detected, the type of evaluation and/or quantitative analyte; on the contrary; any analyte that can be detected by Raman detection; no matter be mark or unlabelled, can be analyzed in the scope of claimed theme.

[0083] in certain embodiments of the invention, one or more " catching " molecule can be attached on the Raman active matrix 240,340 with the mode of covalent bond or the mode of non-covalent bond, with the sensitivity and/or the specificity of the Raman detection that increases analyte.For example, utilize known technology, selected target nucleic acid is had specific oligonucleotide probe (for example be may be attached on the metal surface of matrix 240,340, oligonucleotide probe can be by covalent modification, to comprise the sulfydryl part, it can be incorporated on the matrix 240,340 of washing).Alternately, target protein, peptide or other compounds are had specific antibody, may be attached on the matrix 240.Whether the existence of target analytes, can detect by following method: allowing under the condition that takes place to hybridize with complementary nucleotide sequence, the oligonucleotides that is attached to matrix 240,340 is exposed to sample, washs subsequently, detect the analyte of combination then.In alternate embodiment of the present invention, before being exposed to Raman active matrix 240,340, one or more analytes in the sample can carry out mark with discernible Raman labels, so that the detection of the analyte of combination.Utilize antibody-antigen to, ligand-receptor to or any other is known, the analyte ligand that demonstrates selectivity each other and/or specificity combination is right, similarly method can be used.By handling with all ingredients, remove the analyte and/or the capture molecules of combination, this matrix 240,340 can be recovered and reuse, described disposal route such as wash with acid, water, organic solvent or detergent, chemical treatment, and/or with catabolic enzyme such as exonuclease and/or Protease Treatment.

Embodiment 4: utilize SERS to carry out the detection of rhodamine 6G (R6G)

[0084] Fig. 6 illustrates disclosed method, system and device is used for exemplary analyte, such as the detection and the evaluation of rhodamine 6G (R6G) dye molecule.R6G is the dye molecule that is fully characterized, and it can obtain from the commercial channel of standard, such as Molecular Probes (Eugene, OR).The R6G solution for preparing 114 μ M, use plasma oxidation, dip and decompose the porous silicon matrix that (PODD) silver applies, (SERS) analyzes by surface enhanced Raman spectroscopy, described plasma oxidation, dips and decomposes porous silicon matrix that (PODD) silver applies and press the method for embodiment 1 and 2 and prepare and get.Have average porous porous silicon matrix in various degree, prepare by changing etching condition.R6G solution is diffused in the matrix of PODD silver coating, uses the excitation wavelength of 785nm, analyzes by SERS, according to the method for embodiment 3.Add chemical intensifier (lithium chloride or sodium bromide, the about 1 μ M of concentration), to strengthen Raman signal.

[0085] the SERS emission spectrum that obtains in the porous matrix that the PODD silver with different porositys applies is shown among Fig. 6.Fig. 6 has shown the SERS emission spectrum of 114 μ M R6G, according to the average porosity of correspondence, to the highest curve, is followed successively by 52%, 55%, 65%, 70% and 77% from the curve of below.As shown in Figure 6, along with the increase of average porosity in this scope, the intensity of SERS emission peak increases, and when 77% average porosity, observes the highest intensity.Porosity is increased to more than 77%, will makes porous silicon layer enter unsettled material category, this can cause porous layer to separate from piece silicon (bulk silicon) matrix.When 77% porosity, scanning electron microscopy demonstrates the bore dia (not shown) of the about 32nm of width.

[0086] when 77% average porosity, the intensity of observing Raman emission spectrum increases by 7 orders of magnitude (10 ⁷).This can compare favourably with the enhancing degree of observed about 6 orders of magnitude on coarse silver-colored dull and stereotyped (not shown).Although the SERS emission peak intensity that increases is that the wavelength of emission peak does not change (Fig. 6) as the function of average porosity, this makes the evaluation of R6G not rely on employed average porosity.According to 1.25 * 10 of estimation ^-16The detection capacity that rises, the molecule number of the correspondence of detected rhodamine 6G is about 9 molecules.

[0087] can carry out other research with adenine solution, adenine is at biologically relevant more target molecule.Adenine solution from 90 μ M on the porous silicon matrix of silver coating has detected unique spectral signature (Fig. 7).

Embodiment 5: the Raman detection of nucleotide

Method and apparatus

[0088] in non-restrictive example, the excitation beam of Raman detection device is by titanium: sapphire laser (Mira, Coherent) at near-infrared wavelength place (750-950nm), or produce at 785nm or 830nm place by aluminum gallium arsenide diode laser (PI-ECL series, Process Instruments).Can use pulse laser beam or continuous light beam.Dichronic mirror (holographic notch filters of Kaiser Optical, perhaps, the interference filter of Chroma or Omega Optical) is crossed in the excitation beam transmission, forms the conllinear geometric relationship with the light beam of collecting.Transmitted beam passes micro objective (Nikon LU series), focuses on the Raman active matrix, has target analytes (nucleotide or purine or pyrimidine bases) on this matrix.

[0089] collects with same micro objective from the Raman diffused light of analyte, and reach Raman detector by dichronic mirror.Raman detector comprises condenser lens, spectrograph and array detector.Condenser lens is with the entrance slit of Raman diffused light focusing by spectrograph.Spectrograph (RoperScientific) comprises grating, and it disperses light according to wavelength.The light that disperses is imaged on the array detection instrument (the Back-illuminated Deep-depletion CCD camera of RoperScientific).This array detection instrument is connected to controller circuitry, and it is connected to again and is used for the computing machine that data shift and detector functions is controlled.

[0090] Raman spectroscopy (SERS) that strengthens for the surface, Raman active matrix is made up of metal nanoparticle or the nanostructured that is coated with metal.The silver nano-grain of size in 5 to 200nm scopes prepares with the method for Lee and Meisel (J.Phys.Chem., 86:3391,1982).Alternately, sample is placed on the aluminum matrix under the micro objective.The fixed sample of the data aggregation of discussing below on aluminum matrix.Detected molecule number is determined by the extinction cumulative volume (optical collection volume) of irradiated sample.

[0091] use miniflow attitude passage, single nucleotide also can be detected by SERS.In various embodiments of the present invention, nucleotide can pass through miniflow attitude passage (wide between about 5 and 200 μ m), is transferred on the Raman active matrix.Miniflow attitude passage can make by molding dimethyl silicone polymer (PDMS), it uses (＂ Fabrication of topologically complex three-dimensional microfluidicsystems in PDMS by rapid prototyping such as Anderson, ＂ Anal.Chem.72:3158-3164,2000) disclosed technology.

When [0092] carrying out SERS under having the condition of silver nano-grain, nucleotide, purine or pyrimidine analyte can mix with LiCl (ultimate density 90 μ M) and nano particle (silver atoms ultimate density 0.25M).Use the analyte solution (room temperature analyte solutions) of room temperature, collect the SERS data.

The result

[0093] use top disclosed system, nucleosides list phosphoric acid, purine and pyrimidine are analyzed by SERS.Table 1 has shown the exemplary sensing range of various interested analytes.

The SERS of table 1. nucleosides list phosphoric acid, purine and pyrimidine detects

Analyte	Ultimate density	The number of the molecule that detects
			dAMP	9pM	～1 molecule
Adenine	9pM	～1 molecule
			dGMP	90μM
	6×10 6
		Guanine	909pM		60
dCMP	909μM
			6×10 7
Cytimidine	90nM				6×10 3
		dTMP	9μM				6×10 5
Thymine	90nM				6×10 3

[0094] at adenylic acid, optimal conditions.Determined that LiCl (ultimate density 90 μ M) can provide the best SERS of adenylic acid to detect.By using other alkali halide salts,, the detection of other nucleotide is carried out easily such as NaCl, KCl, RbCl or CsCl.Claimed method is not subjected to the restriction of employed dielectric solution, can understand like this, can use the dielectric solution of other types, such as MgCl ₂, CaCl ₂, NaF, KBr, LiI etc.Those skilled in the art will recognize that, do not demonstrate the dielectric solution of haling graceful signal, will detect the SERS of nucleotide and produce minimum interference.The result shows, above disclosed Raman detection system and method can detect and identify the individual molecule of nucleotide and purine bases.This is to be reported in the Raman detection that single nucleotide level carries out unmarked nucleotide for the first time.

[0095] Fig. 8 has shown the SERS spectrum of the dATP (curve of top) of the dATP solution (curve of below) of 500nM and fluorescein-mark.The dATP-fluorescein available from Roche Applied Science (Indianapolis, IN).The figure illustrates because of strong enhancing with the fluorescein-labelled SERS signal that causes.

[0096] under the instruction of disclosed content, need not excessive experiment, just can make and use all open and claimed method and apparatus herein.Clearly, to one skilled in the art, can the method and apparatus of describing herein be changed, and can not break away from notion, the spirit and scope of theme required for protection.More particularly, some chemically with physiology on relevant reagent can substitute herein the reagent of describing significantly, can obtain same or similar result simultaneously.What all it will be apparent to those skilled in the art substituting and modifying in spirit, scope and the notion that all is considered at theme required for protection.

Claims (30)

1. method comprises:

A) provide by the porous matrix of washing, it comprises the hole with metal level, and described metal level has even metal depth section basically;

B) described matrix is exposed to the sample that comprises one or more analytes;

C) use laser excitation and spectroscopy, detect and/or identify one or more analytes.

2. the semiconductor substrate that the method for claim 1, wherein described matrix is porous.

3. method as claimed in claim 2, wherein, described matrix is selected from the silicon of nanocrystal silicon, monocrystalline silicon, polysilicon, amorphous silicon and laser annealing.

4. the method for claim 1, wherein metal nanoparticle is added to described by the porous matrix of washing.

5. the method for claim 1, wherein described spectroscopy is a Raman spectroscopy.

6. method as claimed in claim 5, wherein, described Raman spectroscopy is surface enhanced Raman spectroscopy (SERS), surface enhanced resonant raman spectroscopy (SERRS), super Raman and/or coherent antistockes Raman spectroscopy art (CARS).

7. the method for claim 1, wherein, described analyte is selected from amino acid, peptide, polypeptide, protein, glycoprotein, lipoprotein, nucleosides, nucleotide, purine, pyrimidine, oligonucleotides, nucleic acid, sugar, carbohydrates, oligosaccharides, polysaccharide, fatty acid, lipid, hormone, metabolin, cell factor, chemotactic factor (CF), acceptor, neurotransmitter, antigen, anaphylactogen, antibody, substrate, co-factor, inhibitor, medicament, medicine, nutrients, prion, toxin, poisonous substance, explosive, pesticide, chemical warfare reagent, the biohazard agent, bacterium, virus, radioactive isotope, vitamin, heterocyclic aromatic compound, carcinogen, mutagen, anesthetic, amphetamine, barbiturate, psychedelic, waste product and pollutant.

8. method as claimed in claim 7, wherein, described analyte is nucleosides, nucleotide, purine, pyrimidine, oligonucleotides, nucleic acid, amino acid, peptide, polypeptide or protein.

9. the method for claim 1, wherein said one or more analytes carry out mark with one or more Raman labels things.

10. method as claimed in claim 9, wherein various analytes carry out mark with diacritic Raman labels thing.

11. the method for claim 1, wherein one or more capture molecules are attached to described by on the porous silicon matrix of washing.

12. method as claimed in claim 11, wherein, described capture molecules is selected from oligonucleotides, nucleic acid, antibody, antibody fragment, antigen, epitope, agglutinin, protein, polypeptide, peptide, receptor protein, part, hormone, vitamin, metabolin, substrate, inhibitor, co-factor, medicine, fit, cell factor and neurotransmitter.

13. method as claimed in claim 8 further is included in the one or more nucleotide of monomolecular horizontal detection, purine or pyrimidine.

14. method as claimed in claim 13, wherein, described nucleotide, purine or pyrimidine are selected from adenine, single AMP, adenosine diphosphate (ADP), atriphos, dAMP, diphosphonic acid desoxyadenossine and deoxyadenosine triphosphate.

15. a device comprises:

A) by the nanocrystalline porous silicon matrix of washing, it comprises the hole with metal level, and described metal level has even metal depth section basically;

B) laser instrument; With

C) Raman detector.

16. device as claimed in claim 15, wherein, described porous silicon is used as sacrifice layer.

17. device as claimed in claim 16, wherein, described sacrifice layer is by metal substitute.

18. device as claimed in claim 17 further comprises metal nanoparticle.

19. device as claimed in claim 15 further comprises the flow cell that can effectively be connected to Raman detector.

20. device as claimed in claim 19, wherein fluid is by the nanocrystalline porous silicon matrix of the washing in described flow cell.

21. device as claimed in claim 15, the wherein said metal level that is comprised homogeneous by the porous silicon matrix of washing.

22. device as claimed in claim 21, wherein, the degree of depth of described metal level is at least 3 microns.

23. a wafer, it comprises by the nanocrystalline porous silicon layer of washing.

24. wafer as claimed in claim 23, wherein, metallic coating comprises silver, gold, platinum, copper and/or aluminium.

25. wafer as claimed in claim 23, wherein, described porous silicon layer by washing comprises porous silicon matrix plasma oxidation, that dip and decompose (PODD).

26. a method comprises:

A) obtain porous silicon matrix and described porous silicon matrix inserted in the miniflow attitude passage;

B) the described stromal surface of oxidation forms silicon dioxide;

C) with the wetting described matrix of metal salt solution; With

D) the described slaine of thermal decomposition is in the oxidized surface formation metal deposition of described matrix.

27. method as claimed in claim 26, wherein, described metal salt solution comprises silver nitrate.

28. method as claimed in claim 26, further comprise described matrix is exposed to the sample that comprises one or more analytes, wherein, described one or more analytes are selected from nucleosides, nucleotide, purine, pyrimidine, oligonucleotides, nucleic acid, amino acid, peptide, polypeptide and protein.

9. method as claimed in claim 28 further is included at least one purine of horizontal detection, pyrimidine or the nucleotide of individual molecule.

30. method as claimed in claim 29, wherein, described individual molecule is selected from adenine, single AMP, adenosine diphosphate (ADP), atriphos, dAMP, diphosphonic acid desoxyadenossine and deoxyadenosine triphosphate.

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